ToxR is a transmembrane transcription factor required by Vibrio cholerae to colonize the human intestine and cause disease, and its operon partner ToxS is required both to protect ToxR from premature degradation as well as for full ToxR activity. ToxR is active as a dimer, and the periplasmic domain has been shown to be required for dimerization. Whereas these results were obtained using microbiological approaches, our laboratory has employed a biochemical approach using purified domains to demonstrate the periplasmic domains of ToxR and ToxS interact. Furthermore, we have shown that bile salts activate ToxR by increasing the interaction between the two periplasmic domains, while at the same time destabilizing the ToxR periplasmic domain. Building off these results, our laboratory has recently purified a ToxR periplasmic domain containing an intra-chain disulfide bond, which appears to be monomeric, in contrast to the previously purified ToxR periplasmic domain, purified without a disulfide bond, which was dimeric. This existence of a stable, monomeric form of ToxR indicates its periplasmic domain is dynamic, undergoing a transition in response to bile salts from an inactive monomer to an active dimer that binds ToxS. In the first aim of this proposal, the dynamics of the ToxR periplasmic domain when exposed to bile salt induced stress will be explored. The recently purified ToxR periplasmic domain with an intra-chain disulfide bond will be used to monitor the transition from monomer to dimer forms. This will be achieved by first determining the structure of the domain by X-ray crystallography or NMR, and then by using NMR to examine if bile salts can bind it specifically. The residues important for binding bile salts and the ToxR dimer interface will be identified by either NMR or by cross-linking mass spectrometry. Once the interface is identified, the amino acids at the interface will be altered, and their effects on ToxR activity will be determined using an OmpT/U reporter assay. ToxS has been presumed to only bind ToxR. However, ToxS is more conserved across Vibrio species than ToxR, indicating it may have other biological roles. Furthermore, alignments of the ToxS periplasmic domain reveals three conserved modules connected by variable linkers. These features suggest the domain is composed of independent subdomains and that ToxS might bind other ligands. To test this hypothesis, we will synthesize all possible subdomain combinations and test their activity in vivo using the OmpT/U reporter as a marker for ToxR activity. The subdomain constructs will be tested for their ability to interact with purified ToxR periplasmic domain and the ToxR/ToxS binding interface will be identified using biophysical methods. Next, the ToxS periplasmic domain will be tested for binding to cyclic-di-peptides and bile salts, small molecules that are reported or known to effect ToxR activity. Finally, potential ToxS binding proteins will be identified by pulling down a tagged-ToxS under various conditions, and identifying the pulled down proteins by mass spectrometry.